ABSTRACT Stem cell technology provides a useful research tool with basic science and clinical applications. The potential use of stem cells for therapy also holds great promise, but there remain several major obstacles. The main obstacles for cochlear applications are the lack of a coherent strategy for large-scale production of true otic cell types and of a tangible approach to introduce and integrate these cells to the cochlear duct. In this proposal, we take steps to address both of these major barriers. In Specific Aim 1, we seek to push the fate of stem cell- derived inner ear organoids toward a cochlear phenotype. Current protocols for generation of inner ear organoids produce clearly defined otic vesicles that develop into organoids with hundreds of vestibular-like sensory hair cells. In normal embryonic development, hedgehog signaling is critical for dorsal-ventral patterning of the otocyst and is essential for cochleogenesis. We will use agonists and antagonists to hedgehog signaling to influence fate specification. Next-generation sequencing will be used to assay hedgehog-dependent signaling in the stem cell-derived otic vesicles, and a combination of morphology and immunohistochemistry will be used to assay cochlear-vestibular specification in the derived organoids. In Specific Aim 2, we will systematically evaluate the survival and integration of otic cells in the cochlea. The main obstacle limiting implantation of exogenous cells into the auditory epithelium is a hostile high-potassium ionic environment and a tight-junction barrier that prevents effective integration. In this aim, we will ?condition? the cochlea by transiently lowering potassium levels and by disrupting cell-cell junctions. The survival and integration of nave stem cells and stem cell-derived otic progenitors will be optimized and maturation examined. The results will pave the way toward a rationale stem cell therapy approach for replacing cells in the inner ear, with applicability to both environmental and hereditary hearing loss.